Analysis of Inflammatory and Homeostatic Roles of Tissue-resident Macrophages in the Progression of Cholesteatoma by RNA-Seq.

BACKGROUND: Tissue-resident macrophages (TRMØs) can act as innate-immune sentinels to protect body against microbe invaders and stimulating materials such as cholesterol crystals in cholesteatoma, as well as to preserve tissue integrity by cleaning unwanted cellular debris. METHODS: TRMØs in the incised middle ear tissues were obtained from the patients with cholesteatoma as an experimental group and the patients without cholesteatoma as a control group. Differential gene expression profiling of TRMØs was conducted between two groups by analyzing GO processes, KEGG and GSEA pathways of inflammation, tissue repair and homeostasis. RESULTS: The current study showed that 145 of 7060 genes were significantly up-regulated (logFC>2 and FDR <0.05) when compared with the patients without cholesteatoma. GO process, GSEA and Cytoscape analysis of the over-expressed genes illustrated the boosted inflammatory and anti-infection functions of TRMØs existed neutrophil function, leukocyte migration, and adaptive immune response involved receptors and signaling pathways. Whereas the homeostasis and repair functions of TRMØs were affected from up-regulated genes, such as over-expressed keratin-13 that helped form the outer keratinising squamous epithelial layer, and over-expressed MMPs that activated the extracellular matrix molecules to promote inflammation and disturb tissue remodeling. Additionally, 74 down-regulated genes (logFC<-2 and FDR <0.05) also affected the homeostasis and repair functions by affecting extracelluar matrix structure and contractile fibres in TRMØs. CONCLUSIONS: The cellular and molecular levels in cholesteatoma is attributable to chronic infection and several disturbed cellular biological processes involving cell integrity and tissue remodeling.

Extensive qPCR analysis reveals altered gene expression in middle ear mucosa from cholesteatoma patients.

The middle ear is a small and hard to reach compartment, limiting the amount of tissue that can be extracted and the possibilities for studying the molecular mechanisms behind diseases like cholesteatoma. In this paper 14 reference gene candidates were evaluated in the middle ear mucosa of cholesteatoma patients and two different control tissues. ACTB and GAPDH were shown to be the optimal genes for the normalisation of target gene expression when investigating middle ear mucosa in multiplex qPCR analysis. Validation of reference genes using c-MYC expression confirmed the suitability of ACTB and GAPDH as reference genes and showed an upregulation of c-MYC in middle ear mucosa during cholesteatoma. The occurrence of participants of the innate immunity, TLR2 and TLR4, were analysed in order to compare healthy middle ear mucosa to cholesteatoma. Analysis of TLR2 and TLR4 showed variable results depending on control tissue used, highlighting the importance of selecting relevant control tissue when investigating causes for disease. It is our belief that a consensus regarding reference genes and control tissue will contribute to the comparability and reproducibility of studies within the field.

Expression of toll-like receptors 2, 4 and nuclear factor kappa B in mucosal lesions of human otitis: pattern and relationship in a clinical immunohistochemical study.

OBJECTIVES: The objectives were to detect and compare the expression of toll-like receptors (TLRs) 2, 4 and nuclear factor kappa B in mucosal lesions of chronic otitis. METHODS: Fifty-five tissue samples obtained from children and adults operated on for otitis were investigated by semiquantitative immunohistochemical methods using polyclonal antibodies for TLR 2, 4 and NFkappaB. Kruskal-Wallis, Mann-Whitney, and Kendall's tau rank correlation tests were used. RESULTS: Stronger expression of TLR2, 4 was found in inflamed mucosa than in the control for children and adults (TLR2: H = 23.86, P < .0011; TLR4: H = 22.80, P < .00 1) (TLR2: H = 17.53, P < .001; TLR4: H = 11.99, P < .001); in cholesteatoma perimatrix compared to tubotympanic lesions in children (TLR2: H = 11.06, P = .004; TLR4: H = 10.61, P = .005) and adults (TLR2: H = 10.73, P = .013; TLR4: H = 9.65, P = .021). No differences were found in NFkB expression (H = 0.042, P = .99). Significant correlations were found for all pairs of molecules in cholesteatoma and tubotympanic mucosa of adults (TLR2, 4: P = .002, P < .001; TLR2-NfkappaB: P = .032, P = .021; TLR4-NFkB: P = .035, P = .0013), only TLR4-NFkappaB in tubotympanic otitis of children (P = .026). CONCLUSIONS: Toll-like receptors 2,4 and NFkB mediate inflammation in cholesteatoma and mucosal lesions oftubotympanic otitis in children and adults. Significant correlations betweenall pairs of molecules in all samples were detected in adults, but only TLR4-NFkappaB in children.

Expression of pattern recognition receptors in cholesteatoma.

Although many immunologic mechanisms have been investigated in studies of the pathogenesis of cholesteatoma, the role of pattern recognition receptors (PRRs) has not been fully determined. Therefore, we assessed innate immune responses in patients with cholesteatoma. We prospectively evaluated 21 patients with acquired cholesteatoma between August 2010 and July 2012. Cholesteatoma specimens were obtained during surgery, and skin from the external meatus of each patient was used as a control. RNA was extracted from these tissue samples, followed by real-time PCR to quantitatively assess the relative expression of toll-like receptors (TLRs), NOD-like receptors (NLRs), retinoic acid-inducible gene (RIG)-I, NO synthase (NOS) and cytokines. The levels of TLR-2, -3, -4, -6, -7, and -10, NOD-2, and IL-1 and -8 mRNAs were significantly higher in the cholesteatoma than in the skin specimens (p < .05). The expression levels of TLR-2 and -3, RIG-I, IL-6, and TNF-α mRNAs were significantly higher in cholesteatomas from women than from men. The levels of TLR-8, NOD-2, IL-12, and TNF-α mRNAs were significantly higher in recurrent than in initial cholesteatoma specimens (p < .05). Hearing level did not correlate with the levels of expression of mRNAs encoding TLRs, NLRs, NOS, RIG-I and related cytokines (p > .05). In conclusion, alterations in innate immunity triggered by PRRs are important in the pathophysiology of cholesteatoma. Gender differences and frequency of surgery may affect the expression of PRRs in cholesteatomas.

Expression of RANKL and proliferation abilities of cultured human middle ear cholesteatoma epithelial cells.

One of the most distinct characteristics of middle ear cholesteatomas is their capacity for bone destruction during the growth process. In this study, we examined the relationship between inflammatory mechanisms and both bone absorption and the proliferation of epithelial cholesteatoma cells. Cultured cholesteatoma epithelial cells were stimulated by lipopolysaccharide (LPS) and dexamethasone (Dex). We found that the expression of receptor activator of NF-kappaB ligand (RANKL) and Ki-67 in cultured cholesteatoma cells was increased by LPS stimulation, indicating that LPS promotes not only bone destruction but also the proliferative activities of these cells. The constitutive expression of RANKL and Ki-67 and the production of IL-6 and IL-8 were significantly inhibited by Dex treatment. Further, Dex significantly suppressed the stimulatory effects of LPS on RANKL and Ki-67 expression and on IL-6 and IL-8 production. Based on results so far, Dex likely exerts a beneficial action against acute inflammation. However, further studies might be required to assess its clinical features.

Expression of tumor necrosis factor-α, interleukin-1α, interleukin-6 and interleukin-10 in chronic otitis media with bone osteolysis.

The purpose of this study was to evaluate the expression of proinflammatory and immunoregulatory cytokines in chronic otitis media. The expression levels of TNF-α, IL-1α, IL-6 and IL-10 were determined by Western blot analysis of tissue samples obtained during ear surgery. The expression levels of TNF-α, IL-1α and IL-6 in cholesteatoma tissues were substantially higher compared to those determined in the granulation tissue. The highest levels of TNF-α, IL-1 and IL-6 proteins were observed in patients with bone destruction. There were no significant differences in the expression of IL-10 levels in cholesteatoma and normal skin, but in the granulation tissue its level was substantially higher. The level of IL-10 in cholesteatoma tissues inversely correlated with the expression of proinflammatory cytokines, the degree of bone destruction and cholesteatoma invasion. Increased expressions of TNF-α, IL-1α and IL-6 in chronic otitis media and a strong positive correlation between these cytokine levels and the degree of bone destruction indicate the destructive behavior of cholesteatoma or granulation tissue.

Immune cell profile in invasive cholesteatomas: preliminary findings.

BACKGROUND: Cholesteatoma consists of keratinizing squamous epithelium, granulation tissue and keratin plugs. The pathogenesis of cholesteatoma may be related to alterations in the stromal immune cell infiltrate. OBJECTIVE: To examine the immunophenotypic characteristics of the immune cell infiltrate in invasive cholesteatomas. MATERIALS AND METHODS: This study included 12 patients with invasive cholesteatomas causing wide bone erosion of the mastoid, middle ear structures, and the bony plates of middle ear cleft. Diagnosis of invasiveness was based on the clinical, radiological and intraoperative findings. Canal wall-down surgical approach was done in all cases to control the disease process. We used the cholesteatomatous tissue specimens to perform immunohistochemical stains for B cells (CD20), T cells (CD3), histiocytes (CD68) and Langerhans' cells (CD1a). Mouse monoclonal antibodies and immunoperoxidase staining methods were used. The results of immunohistology were scored as mean values of positively stained immune cells. The data were compared with findings in 10 specimens of external ear skin (control group). RESULTS: Immunohistochemistry showed highly significant (p<0.00) counts of immune cells in invasive cholesteatomas (CD3: 4.7+/-0.4, CD68:4.6+/-0.5, CD20: 0.8+/-0.1 and CD1a: 0.8 +/-0.1) compared to those in external canal skin (control group: CD3:0.8+/-0.3, CD68: 1.0+/-0.4, CD20: 0.2+/-0.1 and CD1a: 0.1+/-0.1). In cholesteatomas, the predominant of CD3(+) T lymphocytes and CD68(+) cells (histiocytes). Rare CD20(+) cells and CD1a(+) cells (Langerhans' cells) were also observed. CONCLUSIONS: This preliminary study describes the profile of the immune cell infiltrate in invasive cholesteatomas. The numeric dominance of CD3(+) cells and CD68(+) cells suggests that cell-mediated immunity has important role in the development of cholesteatoma and in its autodestructive properties. Further studies are recommended to categorize the T cell subsets in different stages of cholesteatomas.

[Pathophysiology of cholesteatoma]. / La physiopathogénie des cholestéatomes.

OBJECTIVE: To describe the development of cholesteatoma using current knowledge. METHOD: Review of the literature. RESULTS: Cholesteatoma describes a mass of keratin (skin) in the middle ear which consists of a perimatrix and matrix. There are at least three kinds of cholesteatoma in the middle ear one resulting from invagination (retraction's pocket), another from migration and the last one from congenital inclusion. Cholesteatoma needs three successive inflammatory phases, the first leading to a retraction pocket, the second leading to pathology of the epidermis and of the floor of the external auditory canal and the third is the actual phase of cholesteatoma with invasion and middle ear auto-destruction with bone resorption. In this last phase, many factors play a role, collagenasis, osteoclats, cytokines, NO, bacteria and their biofilm and rupture of the retraction pocket. CONCLUSION: Cholesteatoma is an inflammatory disease of the ear caracterised by bone resorption. Current research is starting to appreciate the important role the immune system plays in the pathophysiology of cholesteatoma.

Toll-like receptors 2, 3 and 4 (TLR-2, TLR-3 and TLR-4) are expressed in the microenvironment of human acquired cholesteatoma.

Human toll-like receptors (TLR 1-10) are crucial in the induction and activation of innate immunity in the course of an infection. They are expressed mainly on the cells of the immune system, and also on some epithelia and endothelia. Their ligands so called pathogen associated molecular patterns are abundant on invading microbes. TLR-ligand binding results in cell signal transduction and subsequent production of various proinflammatory cytokines such as IL-1 and TNF-alpha. Acquired cholesteatoma is formed during chronic otitis media in the proportion of cases. It has adverse effects on ear structures, resulting in osteolysis and bone resorption. Its formation and pathogenesis are not fully understood. The current study attempted to search the possible role of TLRs in this somewhat awkward pathological condition. Surgical specimens of human acquired cholesteatoma (n=15) and normal external auditory canal skin (n=5, control tissues) were tested by immunohistochemistry for the presence of TLRs. Three TLRs were examined: TLR-2, TLR-3 and TLR-4. All TLRs tested were demonstrated in matrix (layer of keratinizing epithelium) and perimatrix (granulation tissue) of this inflammatory tumour. Expression of particular TLRs within the keratinizing epithelium was distinct and uneven. In the perimatrix, numerous T (CD3+) cells were seen and relatively few macrophages (CD11c+, HLA-DR+). There was a weak expression of all TLRs on normal (non-inflammatory) skin. Expression of TLR-3 both on the epithelium and some cells within the perimatrix and the presence of T cells may suggest that apart from innate immune responses, mechanisms of adaptive immunity also operate in cholesteatoma. Weak expression of these receptors on normal skin may also suggest the important role of TLRs in the etiopathogenesis of cholesteatoma.

Characterization of patterns of expression of protein kinase C-alpha, -delta, -eta, -gamma and -zeta and their correlations to p53, galectin-3, the retinoic acid receptor-beta and the macrophage migration inhibitory factor (MIF) in human cholesteatomas.

Cholesteatoma is a benign disease characterized by the presence of an unrestrained growth and the accumulation of keratin in the middle ear cavity. Due to roles in cell proliferation, apoptosis and differentiation members of the protein kinase C (PKC) family could be involved in disease progression. This study focuses on the expression of protein kinase C-alpha, -delta, -eta, -gamma and -zeta in the epithelial tissues of 56 human cholesteatomas and their correlations with those of previously characterized distributions of p53, galectin-3, retinoic acid receptor-beta (RARbeta) and macrophage migration inhibitory factor (MIF). We have previously reported this marker set to be correlated with keratinocyte differentiation in human cholesteatomas. Our present data clearly show that the percentage of PKC-alpha (but not PKC-delta, -gamma, -eta and -zeta)-immunopositive cells in epithelial tissue fro recurrent cholesteatomas was significantly higher than in non-recurrent cases. Correlations between the PKC isoenzymes and the biological markers were non-uniform. PKC-alpha (but not PKC-delta, -gamma, -eta and -zeta) expression in epithelial cholesteatoma cells correlated significantly and positively with the percentages of p53-immunopositive cells. The patterns of PKC-alpha and -delta expression, but not of PKC-gamma, -eta and -zeta, correlated significantly and positively with galectin-3 expression. In addition, the correlation levels between the expression of PKC-alpha and -delta and that of galectin-3 varied depending on the infection and recurrence status. Presence of RARbeta correlated significantly (and positively) with the expression of PKC-gamma and -zeta and also in relation to the infection and recurrence status. MIF correlated presence significantly (and positively) with that of the five PKCs under study, depending on whether the cholesteatomas were non-infected or infected as well as non-recurrent or recurrent. In conclusion, the present study suggests that modifications occurring at the level of keratinocyte differentiation in human cholesteatomas involve distinct effectors, to which the activation of PKC-alpha, -delta, -eta, -gamma and -zeta can be added.

[Role of angiogenesis in the pathogenesis of cholesteatoma in adults]. / Znaczenie angiogenezy w patogenezie perlaka ucha srodkowego u doroslych.

The aim of the study was to compare the vessels among cholesteatoma perimatrix, cholesterol granuloma and skin sections and to investigate the localization and expression of CD34 antigen. 14 cholesteatoma specimens were used in this study. Comparative material consisted of skin specimens taken from the retroauricular area during the ear surgical procedure and 4 specimens of cholesterol granuloma collected from children. Monoclonal mouse antibodies against human CD34 were used for immunohistochemistry which was performed using a standard labelled avidin-biotin technique. Automatic imaging analyzing system was used for counting. The skin specimens showed presence of small blood vessels in the subepithelial area and around the glands. The mean number of the vessels in these specimens was 5.44/mm2 and the mean relative proportion of total vessel area/total area of connective tissue: 4.9+/-2.0%. In the specimens of cholesterol granuloma the mean number of vessels was 34.06+/-21.2/mm2 and the mean relative proportion of total vessel area/total area of connective tissue: 22.1+/-3.1%. In cholesteatoma perimatrix, the mean number of microvessels calculated within 1 mm2 of the subepithelial connective tissue was significantly higher (22.79+/-11.2/mm2) and the mean relative proportion of total vessel area/total area of the perimatrix was 12.1+/-2.8%. Angiogenesis appear in the subepithelial connective tissue of cholesteatoma (perimatrix). The process of angiogenesis makes possible and sustains the migration of keratinocytes into the cavity of the middle ear, their increased proliferation and the expansion of cholesteatoma. Therefore, angiogenesis is considered to be one of the destructive features of this disease.

[Immunomorphological evaluation of cholesteatoma]. / Badania nad immunomorfologia perlakowego zapalenia ucha srodkowego.

Molecular and cellular mechanisms in chronic otitis media (COM) with cholesteatoma have not been clearly enough known so far. Investigations on cholesteatoma are focused on its immunological and morphological status. The authors presented the results of immunomorphological evaluation of 31 patients with COM with cholesteatoma, divided into three groups. In the first group there were 4 patients with congenital cholesteatoma, in the second group 19 patient with primary acquired cholesteatoma and in the third group 8 patient with secondary acquired cholesteatoma. Immunohistochemical investigations were performed using antibodies for identification the tenascin, S-100 protein, antigens Ki 67, CD 31, FVIII, HLA-DR, and growth factors TGFbeta1 and EGFR. The immunological activity was assessed for three types and matrix and perimatrix of cholesteatoma. High expression of tenascin was proven in the perimatrix of cholesteatoma and protein S-100 was highly expressed in the cholesteatoma matrix. Ki 67 antigen was seen rarely and mostly was present in basal cells of the matrix. The presence of endothelial cells was proven mainly in the connective and vascular tissue of perimatrix (CD 31, FVIII). The high expression of HLA-DR in matrix confirms the presence of Langerhans cells. The presence of growth factors TGFbeta1 and EGFR was observed in peribasal layer of the epithelium and keratinocytes of cholesteatoma matrix. Activity of immunological processes in COM with cholesteatoma confirms their important role in pathophysiology of chronic otitis media with cholesteatoma.

[Immunological problems in middle ear cholesteatoma]. / Problemy immunologiczne w przewleklym zapaleniu ucha srodkowego z perlakiem.

Regardless of the pathogenesis of congenital and acquired cholesteatoma two questions seem to be fundamental: what is the origin of the keratinizing squamous epithelium, and what influences invasive and hyperproliferative behavior of the epidermis. The authors of this paper advance a hypothesis that the similar mechanism of the skin infection could exist in psoriasis and atopic dermatitis as well as in cholesteatoma. The retraction pocket leads to the loss of the defense mechanisms and development of planctonic bacteria. Direct and indirect signalings of bacterial products, such as endotoxin stimulate the cytokine cascade and therefore the hyperproliferation of the epithelium keratynocytes. The mobilization of the immunological system may an important role in the perimatrix development.

[Expression of osteoclast stimulating and differentiating factors in a murine model of localized inflammatory bone resorption]. / Untersuchungen zur Osteoklastogenese und Knochenresorption im Kalvarienmodell.

BACKGROUND: The pathology associated to cholesteatoma is predominantly a consequence of osteoclast-mediated bone resorption within the middle ear. To assess its pathogenesis a murine model for dermal-implant induced osteolysis was evaluated for the expression of osteoclast stimulating and differentiating factors. METHODS: Mouse calvaria were analysed for the expression of osteoprotegerin ligand (OPGL), osteoprotegerin (OPG) and macrophage-colony stimulating factor (M-CSF) using immunohistochemistry. The detection of osteoclast cell lineage was acquired by immunohistochemistry using markers CD 4, CD 11a, CD 11b, CD 14, CD 51, CD 68 and TRAP. RESULTS: An increased expression of the investigated cytokines M-CSF, OPG and OPGL was demonstrated by immunohistochemistry. The presence of osteoclast precursor cells and mature resorbing osteoclasts was confirmed in time-dependent manner triggered by dermal implantation. CONCLUSIONS: This study reveals the basic events in osteoclast biology in localized inflammatory bone resorption and provides new insights into the comprehension of cholesteatoma-induced bone resorption.

[Markers of epidermal proliferation in middle ear cholesteatoma]. / Markery proliferacji komórkowej w nablonku perlaka ucha srodkowego.

Middle ear cholesteatoma is characterized by the presence in the middle ear cavity of a stratified squamous epithelium with keratin deposits which by constant proliferation leads to extensive bone destruction. The goal of this study was to evaluate, by immunohistochemical study, the expression of epithelial markers of proliferation--Ki-67 and PCNA in the matrix of cholesteatoma. The materials used in this study were 16 acquired cholesteatoma tissues collected from patients in the age 6-17 years during surgery. The specimens from the skin of the external ear canal were employed as the control. In the immunohistochemical specimens staining intensity and distribution of Ki67 and PCNA positive cells in various layers of the epithelium were assessed in three stages scale. The results were compared to the clinical parameters such as--type of cholesteatoma (pars flaccida or tensa), presence of ear discharge, degree of ossicular destruction and involvement of attic and mastoid. In the cholesteatoma matrix Ki-67 and PCNA positive cells were present in basal and suprabasal cell layers and also more superior layers, unlike the control skin were only basal cells show positive staining. The number of positive cells and intensity of staining was also greater in the cholesteatoma matrix than in skin of external auditory meatus. No correlation was found between results of immunohistochemical examination and clinical parameters.

Lipopolysaccharide concentration and bone resorption in cholesteatoma.

HYPOTHESIS: There is a relationship between the local lipopolysaccharide (LPS) concentration in cholesteatoma and local bone resorption in chronic otitis media (COM) with cholesteatoma. BACKGROUND: During the past decade, it has become known that the recruitment of osteoclasts is the main causative factor that induces bone destruction in COM with cholesteatoma. Cellular inflammation factors like cytokines may trigger the osteoclast. Sequel to this, LPS is able to up-regulate cytokines. This makes it of interest to study whether the local LPS concentration is related to bone resorption in cholesteatoma. MATERIALS AND METHODS: Twenty-four cholesteatoma samples and control tissue from COM patients without cholesteatoma were collected. During surgery, the degree of bone resorption was established and classified. Retrospectively, the authors checked whether patients had chronic purulent otorrhea. LPS concentration of the tissue samples was measured by the limulus amebocyte lysate test. The one-way analysis of variance test was used to determine the relation between LPS concentration, otorrhea, and local bone resorption. RESULTS: A significantly higher concentration of LPS was measured in samples from patients with cholesteatoma with bone resorption and otorrhea compared with cholesteatoma without bone resorption and control tissue. There were no significant differences between the LPS levels of the different groups of patients with bone resorption. CONCLUSION: It is suggested that LPS is one of the first factors in the cascade of bone resorption in COM with cholesteatoma.

Gene expression analysis of human middle ear cholesteatoma using complementary DNA arrays.

OBJECTIVE: To identify genes regulated in human cholesteatoma compared with normal skin tissue using complementary DNA arrays. STUDY DESIGN: In vitro analysis. METHODS: Eight cholesteatoma and retroauricular skin samples were obtained from the same patients during surgery. Upregulated and downregulated genes were highlighted using complementary DNA arrays for screening. Reverse transcriptase-polymerase chain reaction and immunohistochemical staining were performed to confirm the results of the complementary DNA array. RESULTS: Twelve genes were found to be induced or upregulated in cholesteatoma compared with skin samples. These included genes involved in cell proliferation and differentiation (eg, calgranulin A, calgranulin B, psoriasin, thymosin beta-10) and cell invasion (eg, cathepsin C, cathepsin D, cathepsin H). Analyses by means of reverse transcription-polymerase chain reaction showed enhanced expression of several genes including calgranulin A, calgranulin B, psoriasin, thymosin beta-10, cathepsin C, cathepsin D, and cathepsin H in cholesteatoma, supporting the findings from the gene array. In addition, it was verified by immunohistochemical analysis that the expressions of Calgranulin A, Calgranulin B, and Cathepsin D were mainly located in cholesteatoma epithelium. CONCLUSION: The observed alteration in gene expression may play a role in various mechanisms of pathogenesis in cholesteatoma.

Osteoclast stimulating and differentiating factors in human cholesteatoma.

OBJECTIVES: To investigate the expression of osteoclast-activating and differentiating factors and to study the occurrence of osteoclast precursor cells and osteoclasts in acquired human cholesteatoma tissue. METHODS: We examined 21 cholesteatoma samples versus 18 normal auditory canal skin specimens for the expression of osteoprotegerin ligand (OPGL), osteoprotegerin (OPG), and macrophage-colony stimulating factor (M-CSF) using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. Immunohistochemistry and computer-assisted microscopy using markers CD4, CD11a, CD11b, CD14, CD51, CD68, and TRAP obtained the detection of osteoclast cell lineage. RESULTS: An increased expression of the investigated cytokines M-CSF, OPG, and OPGL was demonstrated by immunohistochemistry and RT-PCR in cholesteatoma tissue compared with normal external meatal skin. Several CD4-positive cells exhibited a co-expression for OPGL within the perimatrix of cholesteatoma. The presence of osteoclast precursor cells was confirmed in all samples of cholesteatoma tissue. CONCLUSIONS: This study reveals that the number of osteoclast precursor cells is markedly increased in the perimatrix of cholesteatoma tissue. Our results support a concept described for inflammatory arthritis: the inflammation related to cholesteatoma induces bone resorption by release of OPGL from activated T-cells and triggers osteoclastogenesis. This could be a major target for drugs to inhibit osteoclast formation and bone resorption and may be an adjunct in cholesteatoma management.

Air-exposed tissue culture of human middle ear epithelium and meatal epidermis: a method to study the advancing front of cholesteatoma.

The suitability of an air-exposed culture model consisting of a collagen matrix was investigated for constructing an advancing front (AF) of human middle ear epithelium (MEE) and meatal epidermis (ME). Three different culture settings were used: (i ) MEE; (ii) ME; and (iii) AF (MEE + ME). Small tissue biopsies were placed on a fibroblast-populated collagen matrix and grown at the air-liquid interface. After 3 weeks of culture, the MEE and ME outgrowth was differentiated. Light, scanning electron and transmission electron microscopy showed no visible differences compared to native MEE and ME. Cytokeratin 8 and cytokeratin 10 expressions were comparable to the expression seen in the native MEE and ME tissues. Proliferation, which was demonstrated by the expression of Ki-67, was present in the basal layers of cultured MEE and ME. A double layer of cells in which the ME covered the MEE formed the AF. In the AF, the MEE and ME showed the same morphological and immunohistochemical characteristics as in their native tissues. The results of the study show that this in vitro system is a well-defined model system offering the possibility to study the effects of external stimuli on the different epithelia of the AF involved in the pathogenesis of cholesteatoma.